molecules Article
Simultaneous Determination of the Traditional Herbal Formula Ukgansan and the In Vitro Antioxidant Activity of Ferulic Acid as an Active Compound Yu Jin Kim 1,2 ID , Soo-Jin Jeong 1,3, *, Chang-Seob Seo 4, * Jiyeon Yun 1 and Bu-Yeo Kim 1 1
2 3 4
*
ID
, Hye-Sun Lim 1 , Eunjin Sohn 1 ,
Clinical Medicine Division, Korea Institute of Oriental Medicine, Daejeon 34054, Korea;
[email protected] (Y.J.K.);
[email protected] (H.-S.L.);
[email protected] (E.S.);
[email protected] (J.Y.);
[email protected] (B.-Y.K.) College of Pharmacy, Chungnam National University, Daejeon 34134, Korea Korean Medicine Life Science, University of Science & Technology, Daejeon 34113, Korea Herbal Medicine Research Division, Korea Institute of Oriental Medicine, Daejeon 34054, Korea Correspondence:
[email protected] (S.-J.J.);
[email protected] (C.-S.S.); Tel.: +82-42-868-9651 (S.-J.J.); +82-42-868-9361 (C.-S.S.)
Received: 5 June 2018; Accepted: 5 July 2018; Published: 7 July 2018
Abstract: Ukgansan (UGS), a traditional herbal formula composing seven medicinal herbal plants, has been applied in Asian countries for treating neurosis, insomnia, and irritability. Here, the current study performed a simultaneous determination of the seven marker compounds (liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I) using high-performance liquid chromatography (HPLC), to establish quality control of UGS. A 70% ethanol extract of UGS and a mixture of the seven compounds were separated using a C-18 analytical column on a gradient solvent system of 1.0% (v/v) aqueous acetic acid and acetonitrile. Data were recorded at a UV wavelength of 250 nm for glycyrrhizin; 276 nm for liquiritin apioside, liquiritin, and atractylenolide I; and 325 nm for ferulic acid, decursin, and decursinol angelate. The results exhibited high linearity (correlation coefficient (r2 ) ≥ 0.9998) and proper precision (0.38–3.36%), accuracy (95.12–105.12%), and recovery (95.99–104.94%) for the seven marker compounds. The amount of the seven marker compounds at the concentrations from 0.190 to 16.431 mg/g. In addition, the current study evaluated the antioxidant effects of UGS by measuring their scavenging activities against the 2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radicals using in vitro cell-free systems and observed its antioxidant activity. Among the seven components of the UGS extract, ferulic acid dramatically enhanced the scavenging of ABTS and DPPH radicals compared with other compounds. The concentrations of ferulic acid required for a 50% reduction (RC50 ) in ABTS and DPPH radicals were 16.22 µM and 41.21 µM, respectively. Furthermore, UGS extract exerted the neuroprotective effect and blocked the inflammatory response in neuronal hippocampal cells and microglia, respectively. Overall, the established method of HPLC will be valuable for improving the quality control of UGS extract, and ferulic acid may be useful as a potential antioxidant agent. Keywords: Ukgansan; simultaneous determination; antioxidant; ferulic acid; neuroprotection
1. Introduction Ukgansan (UGS), which is also called yokukansan in Japan and Yi-gan san in China, is a traditional Oriental herbal formula composing seven medicinal herbal plants including Uncaria sinensis, Molecules 2018, 23, 1659; doi:10.3390/molecules23071659
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Atractylodes japonica, Poria cocos, Bupleurum falcatum, Angelica gigas, Cnidium officinale, and Glycyrrhiza uralensis. UGS has been utilized to manage various diseases such as neurosis, insomnia, and irritability in children and is a herbal medicine that is approved by the Ministry of Health, Labor and Welfare of Japan. Modern pharmacological and clinical studies have suggested that UGS has the potential to improve insomnia [1,2], borderline personality disorder [3], neuroleptic-induced tardive dyskinesia [4], drug-induced parkinsonism [5], cognitive impairment [6], and the behavioral and psychological symptoms of dementia (BPSD) [7–9]. It is well known that the occurrence of these diseases is closely related to oxidative stress [10–12]. Thus, antioxidant activity should be considered, in addition to the efficacy of therapeutic drugs. The quality of herbal formulas depends on environmental factors, such as harvesting, storing, processing, and formulating methods. Because of their multiple components and the effect of the environment, quality assessment of the major components of herbal formulas is required for investigating their efficacy and safety [13]. Although the identification and simultaneous determination of the components of UGS using high-performance liquid chromatography (HPLC) and HPLC-Q-TOF-MS have been reported [14,15], there are few studies on simultaneous determination of the compounds in UGS using HPLC. Therefore, the current study conducted a simultaneous determination of the marker compounds of UGS for the quality control of UGS using a photodiode array HPLC detector (denoted the HPLC-PDA method), as it is the most popular analytical method. The HPLC-PDA method is a convenient and rapid analytical method to separate and identify the multiple compounds in herbal formulas [16,17]. In this study, simultaneous analysis of seven compounds (liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I) in a UGS extract was performed, and method validation was carried out using the HPLC-PDA method. Moreover, the antioxidant activities of the seven marker compounds were determined using in vitro radical scavenging assays and the biological effects in neuronal cell lines HT22 hippocampal cells and BV-2 microglia. A variety of drugs have shown the neuroprotective activity against damage-induced HT22 cells and the inhibitory effects on neuroinflammation in lipopolysaccharide-stimulated BV-2 cells [18–20]. 2. Results 2.1. Optimization of HPLC Separation HPLC analysis was performed for separating the seven marker compounds (Figure 1) from a 70% ethanol extract of UGS (Table 1). To establish an efficient separation of the seven compounds, various mobile phases were evaluated, including water, acetonitrile, and methanol with trifluoroacetic acid (TFA), acetic acid, and phosphoric acid. The results showed good separation chromatograms using mobile phases consisting of 1.0% (v/v) aqueous acetic acid (A) and acetonitrile (B). The UV wavelength used for quantitative analysis was 250 nm for glycyrrhizin; 276 nm for liquiritin apioside, liquiritin, and atractylenolide I; and 325 nm for ferulic acid, decursin, and decursinol angelate. Using the established methods of HPLC, the seven marker compounds were resolved within 50 min. The retention times of liquiritin apioside, liquiritin, ferulic acid, glycyrrhizin, decursin, decursinol angelate, and atractylenolide I were 12.36, 12.86, 13.95, 33.77, 42.74, 43.25, and 49.99 min, respectively. HPLC chromatograms of the UGS extract in 70% ethanol and the standard compound mixture are shown in Figure 2.
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Figure 1. Chemical structures of the seven marker compounds of UGS. Figure 1. Chemical structures of the seven marker compounds of UGS.
Table 1. Composition of UGS. Table 1. Composition of UGS.
Latin Latin Name Name Uncariae Ramulus Uncariae Ramulus et et Uncus Uncus Atractylodis Rhizoma Rhizoma Alba Atractylodis Alba Poria Sclerotium Sclerotium Poria Bupleuri Radix Radix Bupleuri Angelicae Gigantis Angelicae Gigantis Radix Radix Cnidii Rhizoma Rhizoma Cnidii Glycyrrhizae Radix Radix et Glycyrrhizae et Rhizoma Rhizoma Total amount Total amount Molecules 2018, 23, x FOR PEER REVIEW
Scientific Name Scientific Name
Amount (g)(g) Amount
Origin Origin
Uncaria sinensis Uncaria sinensis Atractylodes japonica Atractylodes japonica Poria cocos Poria cocos Bupleurum falcatum Bupleurum falcatum Angelica gigas Angelica gigas Cnidium officinale Cnidium officinale Glycyrrhiza uralensis Glycyrrhiza uralensis
6 6 8 8 8 8 4 4 6 6 6 6 3 3 41 41
China China China China China China China China Bonghwa, Korea Bonghwa, Korea China China China China
Figure 2. Cont.
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Figure HPLC chromatograms of 70% the 70% ethanol extract of (A) UGS (A) and a standard Figure 2. 2. HPLC chromatograms of the ethanol extract of UGS and a standard mixturemixture (B) at (B) at 250 nm, 276 nm, andand 325325 nm.nm. Liquiritin apioside (1), liquiritin (2), ferulic acid (3), glycyrrhizin (4), 250 nm, 276 nm, Liquiritin apioside (1), liquiritin (2), ferulic acid (3), glycyrrhizin (4), decursin (5),(5), decursinol angelate (6), (6), and and atractylenolide I (7). I (7). decursin decursinol angelate atractylenolide
2.2. Equation, Linearity, andand Limits of Detection (LOD) and Quantification (LOQ)(LOQ) 2.2.Regression Regression Equation, Linearity, Limits of Detection (LOD) and Quantification The linear relationships between the concentrations (x, g/mL) and peak areas (y) of each The linear relationships between the concentrations (x, µg/mL) and peak areas (y) of each compound were expressed by the regression equations (y = ax + b) (Table 2 and Supplementary Figure compound were expressed by the regression equations (y = ax + b) (Table 2 and Supplementary 1). Calibration curves of the marker compounds revealed good linearity (r2 0.9998).2The LODs and Figure 1). Calibration curves of the marker compounds revealed good linearity (r ≥ 0.9998). The LODs LOQs for the tested compounds were 0.015–0.925 and 0.046–2.804 g/mL, respectively.
and LOQs for the tested compounds were 0.015–0.925 and 0.046–2.804 µg/mL, respectively.
Table 2. Linear range, regression equation, correlation coefficients, LODs, and LOQs for compounds.
Compound. Liquiritin apioside Liquiritin Ferulic acid Glycyrrhizin Decursin Decursinol angelate Atractylenolide I a)
Linear Range (µg/mL) 3.125–50 1.5625–25 0.78125–25 6.25–200 12.5–400 6.25–200 0.78125–12.5
Regression Equation (y = ax + b) a) Slope (a)
Intercept (b)
15290 18759 56995 4882.5 30409 35125 62615
4536.9 2614.9 1865.6 1533 77457 38848 1322.9
r2 1.0000 0.9999 1.0000 1.0000 0.9998 0.9998 1.0000
LOD b) LOQ c) (µg/mL) (µg/mL) 0.177 0.052 0.039 0.619 0.925 0.232 0.015
0.537 0.157 0.118 1.876 2.804 0.705 0.046
y = ax + b, y means peak area and x means concentration (µg /mL). b) LOD (Limit of detection): 3.3 × (SD of the response/slope of the calibration curve). c) LOQ (Limit of quantitation): 10 × (SD of the response/slope of the calibration curve).
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2.3. Determination of the Seven Marker Compounds of the UGS Extract The established analytical method using HPLC was used to the simultaneous quantification of the seven marker compounds of UGS extract. The amounts of the seven marker compounds ranged from 0.190 mg/g to 16.431 mg/g. As shown in Table 3, decursin (16.431 mg/g) was the most abundant compound among these seven compounds. Table 3. The content of marker compounds in UGS. Compound
Content (mg/g)
Liquiritin apioside Liquiritin Ferulic acid Glycyrrhizin Decursin Decursinol angelate Atractylenolide I
1.671 ± 0.004 2.014 ± 0.004 0.605 ± 0.002 10.267 ± 0.05 16.431 ± 0.04 7.606 ± 0.002 0.190 ± 0.001
2.4. Precision, Accuracy, and Recovery Precision was represented as the relative standard deviations (RSDs) of the concentrations of marker compounds in mixed standard solutions, and repeated five times at low, middle and high concentration levels. The results for the intra- and inter-day precision and accuracy are shown in Table 4 The intra- and inter-day precision for the seven marker compounds in mixed standard solutions was
